1. Field of the Invention
This invention relates to the field of specific binding assays, particularly immunoassays for determining substances of clinical interest. Specific binding assays are based on the specific interaction between a ligand, i.e., a bindable analyte under determination, and a binding partner therefor, i.e., receptor. Where one of the ligand and its binding partner is a hapten or antigen and the other is a corresponding antibody, the assay is known as an immunoassay.
2. Brief Description of the Prior Art
Many properties of natural cell membranes can be duplicated in simple lipid bilayer systems, referred to as liposomes. One of these properties is lysis. When a vesicular, e.g., liposome, membrane contains an externally accessible antigen, it will react with its corresponding antibody. When the antigen-sensitized liposome reacts with corresponding antibody in the presence of complement, the membrane is irreversibly damaged and can no longer function as an intact selective permeability barrier. This is immunolysis.
The extent of immunolysis has been monitored by using antigen-sensitized liposomes containing any of a wide variety of entrapped marker molecules which are released upon immunolysis. See Hixby, et al., Proc. Nat. Acad. Sci., 64: 290-295 (1969); Kinsky, et al., Biochemistry, 8: 4149-4158 (1969); Kinsky, et al., Biochemistry, 9: 1048 (1970). See also Six, et al., Biochemistry, 13: 4050 (1974); Uemura, et al., J. Biochem, 87: 1221 (1980); and Uemura, et al., J. Immunol. Methods, 53: 221-232 (1982); Kataoka, et al., Biochem. Biophys. Acta, 298: 158-179 (1973); Haga, Biochem, Biophys. Res. Comm., 95: 187-192 (1980); Solomon, et al., Biochem., Biophys. Acta, 455: 332-342 (1976); Tokunaga, et al., FEBS Letters, 106: 85-88 (1979) and Magee, et al., J. Cell Biology, 63: 492-504 (1974).
Specific binding assay systems have been proposed, using a liposome which has been prepared or treated to have surface-bound ligand or ligand analog and a marker or reagent substance enclosed within the vehicle. The remaining reagents for the assay include (1) a binding partner, e.g., antibody, for the ligand and (2) complement to effect lysis of the vesicle upon binding of the binding partner to surface-bound ligand. Generally, see Gregoriadis, et al., Liposomes in Biological Systems, John Wiley & Sons, N.Y. (1980), especially Chapter 12 entitled "Liposomes as Diagnostic Tools".
Also, immunoassay systems have been disclosed in which the use of an enzyme-encapsulating liposome is suggested. Hsia, et al., U.S. Pat. No. 4,235,792 describes a competitive homogeneous immunoassay method which employs immunolysis of an antigen-sensitized liposome containing a marker. Enzymes are among the markers disclosed (col. 6, lines 24-28). Cole U.S. Pat. No. 4,342,826 discloses a specific binding assay using an antigen-sensitized, enzyme-containing liposome. These liposomes are immunospecifically caused to expose enzyme upon binding of corresponding antibody and fixing of active complement. Upon enzyme exposure, the presence or absence of enzymatic activity is detected. Cole emphasizes the advantage of providing a homogeneous system in which enzymic activity is substantially greater upon lysis, e.g., a "signal:noise" ratio of at least 5-10 and preferably above 60.
Interferences in immunoassays can result from a variety of interactions between the sample and the marker. For example, where the marker is an enzyme, interference can result due to the presence of endogeneous enzymes, non-specific binding proteins and enzyme inhibitors in the sample. These interferences can be eliminated by judicious selection of a suitable marker, the addition of blocking agents and/or sample pretreatment steps. Cole describes the separate and independent pretreatment of the sample prior to performance of an assay. However, it is not the purpose of this invention to address these known interferences or techniques for avoiding the same.
Rather, in liposome immunoassays, we have observed a new class of interferences which appear to be due to interactions between the sample and the membrane of the liposome which encapsulates the marker. Certain constituents of the sample appear to bind with the membrane of the liposome and lead to false results which can be higher or lower than is attributable to the antibody-dependent immuno lysis. Whether such false result is higher or lower depends upon the manner in which the interfering substance affects the liposome membrane. The prior art does not provide or suggest means to eliminate these types of interferences.